1. Field of the Invention
The invention relates to compounds having lectinic properties, and to their biological applications.
More particularly, it relates to proteins or polypeptides of the sarcolectin type.
2. Description of the Related Art
It is known that this term has been used to designate the lectins identified in human osteogenic sarcomas or those induced in the hamster (after inoculation with Moloney sarcoma virus) where they are present in large quantities. However, sarcolectins are also found in a wide variety of normal or tumoral tissues in vertebrates. (primates, rodents, fowl).
They may also be detected on the surface of normal or transformed cells of human or animal origin.
Purified preparations of sarcolectins have already been described and their properties reported.
The most regularly characterised physical-chemical properties are:
sensitivity to proteases (trypsin, PRONASE(copyright) (proteolytic enzyme), but also resistance to controlled treatments, under certain conditions, with pepsin;
resistance to temperature variations (xe2x88x9220xc2x0 C. to +100xc2x0 C.);
resistance to pH variations (2 to 8);
resistance to detergents such as SDS and di-thiothreitol;
migration in SDS-PAGE gel of the protein in the molecular weight region of 65-55 kd.
Their biological properties are of three orders (see references (1) to (8) given at the end of the specification, with the other references mentioned below, in the document xe2x80x9cBibliographic referencesxe2x80x9d):
agglutination of normal or transformed cells (activity at cell membrane level). Cytoagglutination may be inhibited due to the affinity of sarcolectins for simple sugars.
stimulation of the growth of human T and B lymphocytes, Daudi lymphoid cells, and cells adhering to the substrate such as L929 murine cells, transformed rat fibroblasts (Fr3T3) and human fibroblasts (FS4). Sarcolectins thus act as promoters of cell growth, of undetermined specificity;
reduction or suppression of the antiviral state pre-established by interferon (IFN) and restoration in the cell of the initial sensitivity to the virus. After antiviral resistance has been established, SCLs inhibit the continuation of the synthesis of interferon-dependent proteins, for example, protein kinase and 2-5A synthetase. Restoration of the initial state depends on the doses of SCL and may be more or less complete. When the cell is restored, it is possible either to stimulate growth by SCLs or by other growth factors, or on the other hand to re-treat the cells with IFN in order to develop antiviral resistance again.
A The invention is based on obtaining highly purified preparations of SCL which have made it possible to develop strategies leading to the isolationof cDNA clones coding for a 55 kd protein, the study of which has revealed unexpected biological properties.
It should be noted that all previous attempts prior to the invention retained the 65 kd protein as the molecule having the biological properties of SCL. During identifications based on electrophoresis on acrylamide gel followed by Western blot, the major band in the 65 kd region had in fact been retained as holding all the biological properties: the 55 kd band is not constantly observed and moreover appears minor in all cases. Now, in a surprising manner, it appears that the 65 kd band corresponds to an artifact which results from the fixation of a few SCL molecules on albumin, but that the molecule possessing sarcolectin-type properties is in reality the 55 kd protein which contains all the genetic information responsible for the biological expression of the molecule.
The object of the invention is, therefore, to provide various products binding to 55 kd SCL, namely in particular, proteins, polypeptides or fragments thereof, DNA sequences coding for these proteins or these polypeptides or fragments thereof, or on the other hand, inhibiting their expression, and antibodies directed against these proteins, polypeptides or fragments thereof.
The term sarcolectin or SCL, as used hereinafter in the specification, will designate without distinction proteins, polypeptides and fragments of these compounds, or their derivatives, as long as they possess lectinic properties as defined according to the invention.
The invention also relates to processes for obtaining these various products.
According to another aspect, the invention also relates to biological applications for these products.
The sequences of nucleotides according to the invention are sequences isolated from their natural environment and are characterised in that they contain at least part of the sequence SEQ ID NO: 1, one or more nucleotides being modified if necessary, it being understood that these sequences are capable of coding for sarcolectins, i.e. proteins, polypeptides or fragments of these compounds, or derivatives, having lectinic properties.
The sequence SEQ ID NO: 1 is given at the end of the specification, with the other sequences mentioned below, in the document entitled xe2x80x9cList of sequencesxe2x80x9d.
The term lectinic properties means the ability of the SCLs to agglutinate normal or transformed cells, their stimulating effect on cell growth and their effect of inhibiting the antiviral effect induced by interferon, under the conditions described in (8).
Such sequences used according to conventional recombinant DNA techniques are capable of coding for proteins or polypeptides, or fragments thereof, having lectinic activity, comprising at least one chain of amino acids as indicated in SEQ ID NO: 1, in which one or more amino acids are modified, if necessary.
These sequences are further characterised in that they are capable of hybridising with at least one fragment of SEQ ID NO: 1 carrying at least part of the genetic information for a sarcolectin. This hybridisation may be carried out under stringent conditions, but also under relaxed conditions as described in (10).
The invention relates in particular to the nucleotide sequence corresponding to the open reading frame running from position 62 to position 1429 in SEQ ID NO: 1.
In this sequence, the domains of the 5xe2x80x2 ends and/or 3xe2x80x2 ends are particularly preferred given that they contain the genetic information for the fragments possessing the said lectinic properties.
From this point of view, the sequence of about 405 bp running from position 62 to 467 in SEQ ID NO: 1, as represented in SEQ ID NO: 2, is quite particularly preferred.
These 5xe2x80x2 and 3xe2x80x2 domains are characterised in that they contain a large proportion of amino acids capable of being phosphorylated, such as serine, threonine and tyrosine.
The various sequences mentioned above may be genome sequences or of the genome type since certain chains of nucleotides may be separated by introns which will be excised to lead to the expression of mature SCLS.
The corresponding mRNA sequences, or the antisense sequences corresponding to the sequences defined above, and the complementary sequences of these various sequences, also come within the scope of the invention.
These various sequences of nucleotides are further characterised in that they contain no DNA of mammals, infectious agents, prions and other materials of natural products.
As indicated above, the invention also relates to the sequences derived from SEQ ID NO: 1.
These derived sequences are obtained by modification, substitution, alteration, mutation or genetic and/or chemical deletion of one or more nucleotides of SEQ ID NO: 1 or of a fragment of SEQ ID NO: 1, it being understood that they possess genetic information for coding for an SCL retaining at least in part the lectinic activity presented by the polypeptide coded by SEQ ID NO: 1, this activity being increased, if necessary.
If desired, these modifications make it possible to adapt the sequences defined above to the expression in a type of vector or host, or to facilitate cell penetration of the coded polypeptide or to increase its activity.
The invention also relates to the expression vectors containing at least one of the nucleotide sequences defined above, under the control of a suitable promoter. It relates in particular to the vectors containing all or part of SEQ ID NO: 1 or SEQ ID NO: 2, or their antisense sequences.
The host cells transfected by these vectors, which contain the recombinant proteins expressed, are also part of the invention.
The cells used for the transfections are, conventionally, eukaryotic or prokaryotic cells or vegetable cells.
The invention also relates to SCLs having lectinic properties as defined above, as novel chemicals.
The invention thus relates to proteins or polypeptides or to their fragments or their derivatives, characterised in that they possess lectinic activity and contain at least part of an amino acid chain in the sequence coded by one of the nucleotide sequences defined above, one or more amino acids being modified, if necessary, as long as this modification does not adversely affect the lectinic properties of the proteins, polypeptides or their fragments, or their derivatives.
These proteins contain no element capable of being confused with albumin.
The invention relates in particular to the SCLs having an amino acid chain in SEQ ID NO: 3 or in SEQ ID NO: 4.
The SCL corresponding to the open reading frame in the sequence SEQ ID NO: 1 contains 469 amino acids and has a molecular weight evaluated at 55 kd.
The sequences of the 5xe2x80x2 and/or 3xe2x80x2 regions of SEQ ID NO: 3, particularly those of the 5xe2x80x2 region corresponding to SEQ ID NO: 4 are essentially responsible for the lectinic function of the SCLs defined above.
Other particularly preferred peptide sequences correspond to peptides having a structure corresponding to that of SEQ ID NO: 4, running from position 41 to 55 in SEQ ID NO: 1 or NO: 4.
It is the peptides having sequence SEQ ID NO: 5 or peptides derived from this sequence and characterized in that they are recognised by monoclonal antibodies capable of reacting with SEQ ID NO: 1, by their own antibodies, but in that they do not react with antibodies directed against the peptide fragment 81 to 95 in SEQ ID NO: 1.
Other preferred peptides sequences correspond to the peptides of SEQ ID NO: 6, having a structure corresponding to that of the sequence running from position 81 to 95 in SEQ ID NO: 1.
It is the peptides having sequence SEQ ID NO: 6 or peptides derived from this sequence, and characterized in that the antibodies directed against this fragment do not recognise the fragment 41-55 above.
It should be remembered that by peptide derivatives ismeant the sequences differing by one or more amino acids (in particular by modification, substitution, deletion) but recognised by the same antibodies as the native sequences.
The peptides SEQ ID NO: 5 and SEQ ID NO: 6, and their derivatives, are capable, when they are fixed on a susceptible cell in vitro, of inhibiting the fixation of the complete molecule and of hindering the expression of its functions, the effects being less pronounced with the peptide SEQ ID NO: 6 or its derivatives.
The SCLs of the invention are further characterised in that they are those obtained by expression, in an appropriate host according to recombinant DNA techniques, of an expression vector containing a DNA sequence of the kind defined above, recovery of the SCL expressed, and purification.
Other SCLs of the invention are fragments or derivatives obtained by modification of the sequences SEQ ID NO: 3 or SEQ ID NO: 4, as long as they retain at least in part the lectinic properties defined above.
As indicated in connection with the nucleotide sequences, the term modification means any mutation, deletion, substitution and/or addition of one or more amino acids, obtained directly at the level of the amino acid chain, or indirectly by modifying the coding nucleotide sequence.
Other SCLs are those obtained by purification from tissue extracts.
The invention relates in particular to the purified SCLs such as those obtained from tissue extracts, by operating as follows:
treatment of the tissue extract containing lectins with pepsin in a controlled manner or at an acid pH, under conditions that make it possible to remove at least the major part of the contaminating proteins, particuarly albumin, whilst retaining the lectinic activity;
chromatography on SEPHACRYL(copyright) (agarose with acrylamide links, a gel filtration media with a fractionation range of 5,000-250,000 daltons for globular proteins) S-200
chromatography on an ion exchange resin such as DEAE-cellulose or CM-TRISACRYL(copyright) (agarose with acrylamide links, a gel filtration media with a fractionation range 200-2,500 daltons);
affinity chromatography using a sugar such as N-acetylneurminic acid as ligand, the purity of the SCLs being verified by reversed-phase HPLC.
The highly purified SCLs are characterised in that they appear to be practically wholly devoid of albumin. They give a single HPLC peak and, after chromatography on SDS gel and denaturation, show three characteristic bands formed from proteins with an estimated size of 65 kd, 55 kd and xe2x89xa614 kd.
The invention relates quite particularly to the SCL characterised by a molecular weight of about 55 kd, as estimated by SDS-PAGE by comparison with polypeptides of defined molecular weight.
It possesses structural and functional properties which distinguish it from the conventional group of intermediate filaments although it is recognised by a type 7 mesothelial anti-cytokeratin monoclonal antibody.
In fact, it takes the form of a monomer, possibly dimer, whilst intermediate filaments are generally polymerised to tetramers or higher polymers. It is an extracellular protein secreted in vivo and in vitro whilst intermediate filaments are intracellular or participate in maintaining the structure.
They possess lectinic properties, that is, they inhibit the actions of IFN, they have the capacity to stimulate cell synthesis and to agglutinate cells. These properties are essentially conferred by the 5xe2x80x2 end, as borne out by digestion by pepsin which is able to suppress the function of stimulating the synthesis of DNA, with retention of the size and sedimentation in SDS-PAGE.
By providing a highly purified SCL, the invention makes it possible on the one hand to characterise the molecule and on the other hand to obtain sufficiently pure antibodies to characterise the antigen structure of the SCLs.
The invention also relates to processes for obtaining nucleotide sequences and the SCLs defined above.
In order to obtain the nucleotide sequences, it is possible to operate by means of synthesis according to conventional techniques. As a variant, particularly for obtaining at least a part of the sequences of the SEQ ID NO: 1 or SEQ ID NO: 2 type, a bank of cDNA is screened with the aid of specific probes such as antibodies directed against the purified 55 kd protein mentioned above and described in more detail in the examples, or mRNA. The sequences of interest are isolated, if necessary modified as desired, according to the applications envisaged, and undergo one or more purification treatments.
In order to obtain the SCLs of the invention, these sequences are advantageously introduced into an appropriate expression vector, under the control of a promoter, for the purpose of transfection of a host cell.
By applying the conditions required to obtain the expression of the SCL of interest with lectinic activity, the latter is synthesised and, after lysis of the cells or simply after secretion, it is recovered in the recombinant form and undergoes at least one purification stage.
For the production of SCLs, prokaryote systems such as bacteria will be used, or eukaryotes such as insect cells (Baculovirus system), or yeasts, advantageously those available commercially. It is also possible to use animal cells such as CHO hamster cells, or primate cells transfected with the appropriate gene.
The SCLs of the invention may also be obtained by synthesis.
The processes described above allow the purification of the SCL molecules whilst retaining their biological properties.
The invention relates in particular to a process for obtaining SCL with a high degree of purity from tissue extracts, characterised in that it comprises stages for treating the tissue extract containing lectins with pepsin in a controlled manner or at an acid pH, followed by chromatography under conditions whereby at least the majority of the contaminating proteins, quite particularly albumin, can be removed whilst retaining the lectinic activity.
These chromatography stages comprise:
running the pre-treated tissue extract through SEPHACRYL(copyright) (agarose with acrylamide links, a gel filtration media with a fractionation range of 5,000-250,000 daltons for globular proteins) S-200;
recovering the fraction containing most of the lectinic activity as measured by the cell agglutination test;
running this fraction through ion exchange resins, particularly DEAE-cellulose and CM-TRISACRYL(copyright) (agarose with acrylamide links, a gel filtration media with a fractionation range of 200-2,500 daltons);
running the fraction containing most of the lectinic activity through a column containing a sugar such as N-acetylneuraminic acid as ligand.
This succession of stages provides a technical solution to the problem of separating the contaminating albumin and makes it possible to remove it in spite of the size, which is similar to that of SCL, certain physical-chemical properties which are similar to those of SCL, and possible interactions between albumin and SCL.
The tissue extract treatment stage is carried out with the aid of pepsin, operating under controlled conditions. The respective concentrations are of the order of 4 to 8%, preferably about 6% for the tissue extract, and 0.5 to 2 mg/ml, preferably about 1 mg/ml for a pepsin having an activity of the order of 2500 to 2700 units/mg.
Tissue extract treatment conditions which have proved to be advantageous involve incubation of the reaction mixture at about 37xc2x0 C., pH 2, for about 1 h 30 to 2 h 30, in particular for 2 hours.
The enzyme activity is then stopped by raising the pH to a value close to neutrality.
The extract thus treated undergoes a succession of chromatographic stages.
1xe2x80x94Chromatography on SEPHACRYL(copyright) (agarose with acrylamide links, a gel filtration media with a fractionation range of 5,000-250,000 daltons for globular proteins)
Preferably, this extract is centrifuged beforehand and the supernatant undergoes filtration over a SEPHACRYL(copyright) (agarose with acrylamide links, a gel filtration media with a fractionation range of 5,000-250,000 daltons for globular proteins) S-200 gel. The active fractions are recovered by elution with a buffer solution such as PBS in a quantity of 15 to 25 ml/h.
2xe2x80x94Chromatography on DEAE-cellulose The fractions recovered are pooled and the fractions with the maximum lectinic activity undergo chromatography on DEAE-cellulose which as been swollen beforehand and equilibrated with a buffer solution with a pH around neutrality, particularly a pH of the order of 7.6.
Elution is carried out with the buffer solution to which has been added a salt with a molarity ranging from 0 to 0.5 M representing a linear gradient of pH 7.6 to 4.0. A satisfactory rate of elution is of the order of 20 ml/h.
3xe2x80x94Chromatogra on CM-TRISACRYL(copyright) (agarose with acrylamide links, a gel filtration media with a franctionation range of 200-2,500 daltons)
The active fractions recovered are chromatographed on CM-TRISACRYL(copyright) (agarose with acrylamide links, a gel filtration media with a fractionation range of 200-2,500 daltons) equilibrated in a buffer of pH 4.2, particularly a 0.04 M sodium acetate buffer.
Preferably, the active fractions are dialysed beforehand against the acetate buffer, then placed on the CM-trisacryl(copyright) (agarose with acrylamide links, a gel filtration media with a fractionation range of 200-2,500 daltons) column, and rinsed with the same buffer.
In order to remove the albumin, a first buffer of pH 5, 0.1 M is used, which operation may be carried out in 1 hour. The use of a second buffer, pH 4.2, 1 M, allows the majority of the SCLs to be eluted. This operation may be carried out within about 20 minutes. Before bringing the active fractions into contact with a sugar, it is advantageous to carry out dialysis to remove the dialysable molecules using a 0.01 M Na phosphate buffer, pH 7.2.
4xe2x80x94Affinity chromatography using a sugar as ligand:
The sugar constitutes the ligand of an agarose gel affinity chromatography column, particularly hexamethylene-diamine polyacrylamide-agarose.
The gel is first washed with a 0.5 M buffer, for example NaCl, then with distilled water and finally centrifuged at low speed so as to remove non-adsorbed substances. A sugar solution, pH 4, for example 0.1 M N-acetylneuraminic acid, is added to the gel followed by a solution of an agent such as a carbodiamide.
The pH is advantageously kept at a value of the order of 4.5 and 5 at ambient temperature for about 1 hour, then gentle agitation is carried out for 10 to 15 hours.
The gel is washed several times to remove non-retained impurities.
For example, washing takes place with 1 M NaCl, then with 0.1 M acetic acid, and finally twice-distilled water.
The column packed with this preparation, is equilibrated to pH 7.2. The use of a 0.01 M sodium phosphate buffer has proved to be suitable.
The active sample of the previous stage is placed on the column and is advantageously equilibrated beforehand with the phosphate buffer.
After the column has been rinsed with this buffer, elution is carried out with at least two buffers, one for eluting the SCL and the other for removing the other proteins and regenerating the column.
The first buffer (I) is advantageously composed of a 0.01 M solution of sodium phosphate pH 7.2 containing 0.15 M NaCl. It leads to a peak containing the majority of the SCL. The second buffer (II) also contains ethylene glycol, particularly in a quantity of about 40 to 60%, preferably 50%. It leads to another peak containing the majority of the impurities.
The column is then rinsed with the sodium phosphate buffer.
5xe2x80x94Verifying the purity
The SCL recovered is analysed by HPLC using a conventional water/acetonitrile/trifluoroacetic acid system and the fraction corresponding to the main peak is then analysed by SDS-PAGE.
The invention thus gives methods of isolating very high purity products and of providing a product from which practically all the albumin has been removed.
The study of the purified SCLS thus obtained and of the SCLs corresponding to the expression products of the nucleotide sequences defined above shows that they possess lectinic properties of great interest.
1. These SCLs do not act directly on interferons but inhibit the synthesis of the secondary effector proteins induced by interferons.
The number of these effector proteins induced varies from one interferon isoform to another Moreover, the same molecular form does not necessarily induce the same effector proteins from one cell to another. By inhibiting the synthesis of all the IFN-dependent secondary proteins, the SCLs restore the cell to its initial state. Consequently, the cells regain their capacity to respond to growth stimuli which would otherwise be inhibited by the interferons.
2. The direct stimulation of growth is obtained in the absence of serum and affects a large number of cells, whether or not they are immunocompetent. This stimulation is obtained without a retro-inhibition effect which results in the development of the state whereby the cells are unaffected by repeated inductions of this substance.
Consequently, unlike conventional immunostimulants, the SCLs of the invention may be administered. repeatedly, if necessary in association with specific growth factors with which they may act synergistically. An example is interleukin-2 (Il 2) which is unable to stimulate the proliferation of T lymphocytes unless these lymphocytes are activated beforehand by a lectin or another antigen. Sarcolectin could thus act as a physiological activator of the receptors of Il 2. In other examples, sarcolectin will be associated with various interleukins or growth factors with a view to targeted amplification of growth due to the resulting synergy.
3. The mechanism of carcinogenesis seems to be clarified at least in part. It is generally assumed that the malignant transformation is the result of a process which, in progressive stages starting from benign proliferation, ends in the selection of highly carcinogenic cells. The protooncogenes are genes which are involved in the normal process of proliferation either as the growth fact or as the corresponding receptor, or by taking part in the metabolic chain involved in the growth process. The SCLs of the invention, more particularly the SCLs corresponding to SEQ ID NO: 3 or SEQ ID NO: 4 or the derived sequences are involved in initiating the synthesis of the cell DNA preparing the action of specific growth factors. In particular, the 55 kd SCL described above is a constitutive glycoprotein which is secreted in the extracellular medium and stimulates growth in a non-specific manner. As the effect of sarcolectins and growth factors is additive, it may be regarded as a co-oncogene, on the one hand due to its own effect on cell proliferation and its synergy with growth factors, and on the other hand due to its inhibiting effect on the anti-proliferative functions of interferons. As with all lectins, the biological functions of sarcolectin are inhibited by specific sugars.
In view of the properties reported above, the invention relates more particularly to the following applications of SCLs.
The invention therefore relates to their use as growth co-factors, for contributing to tissue growth and in particular for contributing to the regeneration of damaged tissues and to the acceleration of wound healing.
In this respect, they constitute highly effective therapeutic agents of local or general application.
As growth factors, they may be used for cell cultures in vitro.
In this respect, the particularly preferred products are constituted by human recombinant SCLs.
Indeed, when lymphocytes freshly removed from a healthy individual are cultivated, it is possible to stimulate their proliferation in the presence of interleukin-2.
After successive runs in the H1 medium without serum, the cells replicate in the presence of only Il2. However, the only protein secreted in the medium is SCL rn in a dimer form.
In H9 continuous T lines, cellular multiplication is assured by the SCL produced by the cells and secreted. In fact, in both cases, it is the only major protein detected in the medium by SDS-PAGE and Western blotting.
Tests carried out with the addition of SCL in an appropriate quantity show its favourable effect on cell growth.
The invention therefore also relates to the use of SCLs as therapeutic agents for stimulating the immune system, particularly as a stimulant of specific immunity; if necessary, the SCLs are used in association with an antigen, for example, with a growth factor such as interleukin.
As physiological activators of the proliferation of T lymphocytes, SCLs are therefore able to activate the synthesis of receptors for interleukins (Ils), particularly Il2. The continuous expression of SCLs in the cell appears to inhibit the state whereby the cells are unaffected by repeated inductions of interferon, leading to the continuous production of interferon on each induction, though the cells are incapable of expressing IFN functions.
The invention relates in particular to their use in treatments with interferon by taking advantage of their effect of inhibiting the synthesis of secondary effector proteins in such a way as to restore the cell to its initial state and restore its normal sensitivity to interferon.
The SCLs or their inhibitors are thus advantageously used in protocols for the repeated administration of IFN, for treating pathological states of infectious origin, for example, during the final stages of AIDS infections or during auto-immune diseases such as lupus erythematosus.
In certain competent cells, in particular those of PBL, the addition of recombinant SCL causes an agglutination and the synthesis of interferon of type 1 or 2.
The SCLs of the invention may also be used as vaccination adjuvants due to their ability to increase the proliferation of immunocompetent cells.
According to another aspect, the invention relates to the use of the SCLs of the invention as tools for identifying compounds that inhibit natural sarcolectins.
In particular, the invention relates to the use of SCLs for selecting compounds that inhibit their lectinic activity by competition, the antibodies or sugars, or also antagonists such as butyric amino acids or other butyroids.
In the different therapeutic uses mentioned above, the SCLs are, if necessay, fixed to albumin with a view to stabilization, to produce a delayed-action vehicle or to facilitate their diffusion in tissues and the expression of their functions.
The medicinal products developed from the inhibitory compounds mentioned above, which contain efficacious quantities of these compounds for obtaining the inhibition of interest, in association with pharmaceutical excipients, also fall within the scope of the invention.
Inhibitory sugars are specific sugars present on the cell membrane. They are simple or compound sugars such as N-acetylgalactosamine, sodium galacturonate, acetylated sugars such as N-acetylneuraminic acid, alpha or beta lactose, galactose, neuramine lactose
These sugars, by fixing on the cell membrane, interfere with the fixation of SCL. On the basis of these observations, complex butyric derivatives can be obtained by fixing galactose, lactose, NANA, glucosamine, galactosamine, N-acetyl galacturonic acid on these butyric derivatives or on other butyric derivatives such as esters containing, for example, octal butyrate or PEG butyrate. (PEG=polyethylene glycol).
On the whole, the majority of these inhibitors indirectly increase the expression of induced interferons, increase the expression of endogenous interferon during different phases of normal growth or the process of tumorigenesis.
The butyroids can be used as growth inhibitors; they produce an interferon-like effect, but by different mechanisms. Their effect is not inhibited directly by SCLs, but acts as an antagonist effect of SCLs by different mechanisms.
The butyric derivatives formed from hydrophobic-hydrophilic amino acids such as 1-valine t-butyl ester, by their hydrophilic functions, fix to the cells and by their hydrophobic functions, maintain the molecules in contact with the membrane.
The butyric amino acids include alpha aminobutyric acid, alpha aminoisobutyric acid and gamma aminobutyric acid (GABA). These compounds have a high affinity in particular for the transformed cells and inhibit graft taking of Sarcomes TG180 cells (or of other cells) in mice.
As a result of these inhibitors or antagonists, excessive or continuous anti-physiological production of interferons may be re-equilibrated (by inhibiting the action of SCLs or by opposing their effects using antagonists) in order to combat immune disorders induced by certain chronic viral infections (HIV) or immune diseases.
The inhibitory compounds are capable of increasing considerably the antiviral resistance induced by IFN and are used to advantage in protocols including treatments with IFNS. They may also be used in anti-cancer therapies.
The invention relates in particular to the use of these inhibitors in such treatments in association with immunomodulators such as corynebacterium parvum that SCL may replace if there is no constituent production of SCL.
Other products capable of inhibiting the activity of the SCLs of the invention by competition are composed of antibodies. These polyclonal or monoclonal antibodies directed against the SCLs of the invention are produced advantageously according to conventional methods. As novel products, they are a further object of the invention.
In therapeutics, the anti-SCL antibodies of the invention are particularly valuable agents for inhibiting the effects of the SCLs produced in excess in pathological states such as cancers, chronic viral or auto-immune diseases.
The medicinal products developed from these antibodies are characterised in that they contain an efficacious quantity of these antibodies for the applications envisaged, in association with an inert pharmaceutical vehicle.
These medicinal products are particularly suitable for anti-tumour treatments.
In diagnostics, these antibodies may be used for all the immunological reactions, particularly ELISA and Western blots, and make it possible to determine qualitatively and quantitatively the presence of SCL in a biological extract taken from a patient.
The invention thus relates to a method for detecting in vitro SCLs, and more particularly the 55 kd SCL as purified according to the methods described above, or corresponding to the protein expressed by SEQ ID NO: 1, or obtained by synthesis.
Said method comprises:
bringing a biological sample to be analysed originating from a patient or cells into contact with an anti-SCL antibody preparation or a Fab fragment immobilised on a solid support under suitable conditions for the production of an antigen-antibody complex with the SCLs if they are present in the sample or cells, then,
detecting the formation of such a complex of the antigen-antibody type, advantageously by operating according to usual methods.
For example, cytofluorometry methods are used.
This method of detection makes it possible to detect with great sensitivity and at high speed the presence of SCL in the sample tested, and to detect any reaction of an antigen-antibody type.
The invention also relates to a kit that can be used to carry out this detection. This kit is characterised in that it comprises:
a suitable solid phase acting as a support,
an anti-SCL antibody preparation or Fab fragments, free or immobilised,
buffer solutions and suitable reagents for the immunological reactions and for the detection reactions.
The antibodies used in these methods and kits are advantageously antibodies directed against the peptides SEQ ID NO: 5 defined above or their derivatives.
As a variant, the detection relates to the presence of genes coding for the SCLs and comprises
carrying out the stage of bringing the biological sample to be analysed or cells originating from a patient into contact with a probe as defined above under suitable conditions for the production of a hybridisation complex, if the genes coding for the SCLs are present in the sample or cells,
detecting the hybridisation complex and quantifying the expression of the SCLs by these genes.
The invention also relates to a kit that may be used in this method and comprises said probes and the buffer solutions and useful reagents for performing the hybridisation reaction.
Other inhibitors of the SCLs of the invention are composed of the antisense nucleotide sequences defined above. These antisense sequences are able to block the expression of SCL, for example, in the case of osteogenic sarcomas.
Other applications for the SCLs according to the invention are based on their ability to agglutinate cells and their affinity for simple sugars.
These properties are turned to good account in diagnostics or therapeutics.
Other characteristics and advantages of the invention will be given in the examples that follow.